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PSYC 370 > MT1 > Flashcards

MT1 Flashcards

1
Q

prototypical vertebrate neuron

  • 9 parts
  • defining characteristic
A
  • dendrites
  • cell body + nucleus
  • axon hillock
  • axon + myelin + nodes of Ranvier
  • presynaptic + postsynaptic terminals

Defining characteristic: they fire APs

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2
Q

types of cells

  • unipolar
  • bipolar
  • pseudo-unipolar cell

multipolar cells (3)

  • motor neuron
  • pyramidal cell
  • purkinje cell
A
  1. unipolar: no dendrites attached to cell body (only axon)
  2. bipolar: axon + dendrite attached to cell body
  3. Pseudo-unipolar: in between 2 neurons but not connected –> can bypass cell body (go from dendrite to axon) –> for reflexes (in the peripheral NS get a lot of inputs –> connections with many other cells –> responsible for coordination
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3
Q

neuron functions

A

input: dendrites receive signal
integrative: cell body processes/combines signal –> if strong enough AP generated
conductive: AP conducts along axon
output: axon button releases NTs to next cell (synapse)

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4
Q
  • sensory
  • motor
  • neuroendorcrine
A

sensory neurons: receives input from the outside world, not another neuron
motor neurons: receives input from other neurons (many dendrites bc they receive a lot of info) –> output to muscles (Ach causes muscles to contract)
neuroendocrine: project into body/brain –> release NTs or peptides –> get fight or flight response going

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5
Q

problems studying circuits

  • connectome
  • stretch reflex circuit
A
  • we dont know how neurons interact at a circuit based level
  • if we know the connectome (synaptic connectivity of all neurons) we can infer the circuits function (based on knowledge of how indiv parts work)

stretch reflex circuit:

  1. we know sensory neurons can sense stretch –> AP generated in 1D
  2. we know sensory neurons release excitatory NTs
  3. reflex = muscle –> pass cell body –> spinal cord
  4. cell in spinal cord gets excited by NTs –> signal propagated in 1D to the end of axon –> back to muscle

therefore, the function of this circuit is to sense stretch and send message to counteract the stretch

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6
Q

visualizing neurons

  • unprocessed brain slice
  • indiv neuron
  • cortical neurons
A

unprocessed: brain is translucent –> useless –> cant see anything with just a light microscope

indiv neuron: single out one and stain red –> give nuclear stain to rest to make bg –> look at multiple indiv neurons and try to see if they’re unique –> see what they’re doing (function)

cortical neurons: come from brain –> carry sensory info (storing info? hippocampus stores memory)

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7
Q

complementary approaches

A

options: combine to see diff aspects –> each has advantages/disadvantages –> depends on expt
- dead tissue vs live tissue: some only work on dead tissue –> want to see changes over time wont work (need live tissue) –> if you want to see disease model (end product) dead tissue is fine
- cell type or molecule presence: can track proteins over time
- cheap vs expensive; low vs high resolution: electron microscope SO EXPENSIVE compared to light microscope, but much higher res
- easy vs hard: very difficult to raise transgenic mice with cells that glow in the brain

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8
Q 
Methods for visualizing neurons
Method 1: golgi stain
- stain with what
- con
- pro
- study
A

use silver nitrate to wash tissue –> randomly fills some neurons –> very clear neuroanatomy (random tho)

  • dead tissue only
  • relatively easy
  • can study synaptic pruning, schizophrenia (patients have fewer spines in PFC –> overpruning)
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9
Q 
Methods for visualizing neurons
Method 2: dye filling neurons
- target
- how to fill?
- study what?
- correlates with?
A

target living or dead cells –> use micropipet to inject fluorescent dye –> able to see neuroanatomy

  • put electrode in pipet (if you can see pipet poking out you know it is dye filling method!!) –> can measure electrophysiology
  • autism = underpruning (more spines = more synapses)
  • can correlate spines w electro activity –> see if spines are important for synaptic activity
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10
Q 
Methods for visualizing neurons
Method 3: immunohistochemistry
- difference from methods 1/2
- procedure
- advantages/disadvantages
- AD
A
  • labelling proteins not staining cell
  • takes advantage of an animals ability to make antibodies
  • can make antibodies for protein (antigen) –> inject into animal
  • immune cells produce those antibodies –> label with fluorescent tag attached to antibody
  • can label multiple proteins and get diff combos of colours –> multiple cell types in same tissue –> localization
  • dead tissue; relatively cheap and easy
  • shows that first sign of AD is loss of synaptic proteins
  • wash with antibodies –> if you get fluorescence it means the protein is there, but for AD post mortem patients we see its not there
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11
Q

immunohistochemistry examples

  • DAPI
  • doublecourtin
  • calretinin
A

DAPI: stains all cells –> marks cell body

doublecortin: cells extend dendrites toward targets –> this protein is important for this signaling
calretinin: inhibitory interneuron –> hypothesis that they play some part in the expression of immature neurons and how they grow

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12
Q

Methods for visualizing neurons
Method 4: genetically-encoded fluorescent proteins
- genome
- promoter regions

A
  • All cells in body are made of same DNA –> differential expression of diff proteins —> carry out diff functions —> unique cell (distinct neuron types, tissues, regions, etc)
  • On DNA there are promoter regions –> can artificially create promoter region and inject into cell of interest
    —> can make proteins we want
  • Make GFP code after promoter region —> can see where the cells are bc GFP is attached to (eg doublecortin)
  • Can target protein —> check if cell has that protein (if we know it does, can track the cell bc of GFP)
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13
Q

GFP

  • where did it come from?
  • emission vs excitation spectra
A

isolated from jellyfish
on a GFP emission spectrum –> most intense light is green area (~509nm)
- dotted blue line = excitation spectrum –> the light you need to shine in order to cause GFP to emit light (~487nm)

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14
Q

genetic constructs

  • 2 options
  • advantages/disadvantages
  • genetic modulation
  • challenge
A
  1. can create transgenic mice –> insert DNA into embryo –> expresses GFP
    - costly to set up, cheap and efficient once you have it (can just keep breeding transgenic mice, or order mice from breeders)
  2. can insert viral vector into cell –> engineered, less toxic and more controlled than reg virus
    - can label genetically-identified cells, or label modified cells (did DNA insertion work?)

genetic modulation: eg. if you’re trying to stop production of a sp protein you can put GFP in so you can look at animal and see if it worked (labelling technique)

Challenge: variable gene expression

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15
Q

choosing the right promoter

  • why critical
  • CAG
  • transplants
  • drawback
A
  • critical to choose good promoter (bad promoter = silent region = no GFP made)
  • can get GFP everywhere using CAG, a promoter active in all cells (actin is in all cells) –> will see activation anywhere with skin
  • can use this to see if donor/transplants worked –> eg. liver transplant will show GFP if cells survived

drawback: no specificity –> not good for indiv neurons
- -> more used for “did my method work”

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16
Q

Other promoters:

  • thy-1 promoter
  • L7 promoter
  • doublecortin
  • Iba-1
  • GFAP
A

Thy-1 promoter: active in a fraction of all types of neurons –> can do in-vivo imaging (live tissue)
- drawback: only in neurons expressing thy-1 –> only a subset show –> don’t know if they’re the same as their neighboring ​cells or not

L7 promoter: active (produces GFP) only in cerebellar purkinje neurons

doublecortin promoter: only in immature neurons

Iba-1 promoter: only in microglia –> able to see curve of hippocampus

GFAP promoter: only in astrocytes –> supportive in the brain

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17
Q

expressing FPs with viruses

  • transgenic animals
  • targets
  • retrovirus
  • pros and cons
A
  • inject engineered viral vectors into a specific brain region
  • does not require transgenic animals to obtain genetically modified cells (faster, cheaper)
  • can target diff cell types with diff virus types (neurons vs glia, dividing vs non-dividing)

eg. retrovirus: only affects dividing cells (want a cell that is a day or 2 old) –> brand new cell
- engineered to do 1 round of replication and stop (safe)
- need to do surgery to locate area of brain you want to inject –> invasive!

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18
Q

clarity technique

  • effect
  • advantage
  • disadvantage
  • thy-1
A

brain clearing: clear membranes/lipids/fats which scatter light –> light can penetrate deeper, emitted light will be captured without scattering

  • allows imaging of FPs in larger blocks of tissue –> don’t need to cut thin slices –> can see one axon from start to finish
  • dead tissue only –> neurons are not static, so if you wanted to see changes over time you would need live tissue
  • Thy-1 lights the entire brain with fl –> ionizing liquid will ionize micelles –> leaves proteins and carbs behind (which is what you want)
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19
Q

confocal microscopy

- fluorescent vs confocal

A

fluorescent: all light from tissue is reflecte to eye piece
confocal: shines laser –> pinhole eliminates out of focus light –> single focal plane –> reconstructs images from diff focal planes
- difficult to see middle tho due to blurring from top/bottom layers

20
Q

photon microscope

  • spine density
  • how to check if spines have synapses?
A

can go deeper into skin –> seeing how brain changed in response to stimuli
- spine density does not change, but location does –> in response to experience, more transient spines occur (taking away exp = need more spines –> randomly form, if theres no activity they go away)

how to check if spines have synapses?

  • immunohistochemistry: light up proteins in pre and post synaptic membranes
  • electron microscopy: check spines labelled with GFP and see if there are synapses
21
Q 
Methods for visualizing neurons
Method 5: Electron microscopy
- how it works
- stain
- advantage
- disadvantages
- immunohistochemistry
procedure (3 steps)
A
  • shoots electrons through structure
  • must stain with heavy metal stain
  • best resolution: clearest images of small details
  • expensive, time consuming, need vacuum and stable env
  • immunohistochemistry: wherever you have GFP you will get stain for electron microscopy –> if its too thick you cant see indiv neurons

procedure:

  1. tissue embedded in resin
  2. cut into 60 nm thick sections (blade of diamond or glass)
  3. imaged on electron microscope –> stack images to make 3D model
    - very dangerous –> any vibrations can mess up
22
Q

fundamental neuronal signals (2)

A
  • action potentials - electrical
  • synaptic transmission - chemical

a stimulus from the outside world is converted into neural signals (electrical) –> decisions are made –> behaviour is executed (circuit)

23
Q

membrane potential

  • equation
  • resting potential
A

Vm = Vin - Vout

Vm = membrane

  • inside is negative, outside is positive (due to characteristics and concentrations of ions
  • RMP = -65mv
24
Q

glial cell RMP

  • dictated by?
  • concentration gradient
  • electrical gradient caused by?
  • when are 2 forces equal?
A
  • dictated by [K+]
  • [K+] much higher inside than outside
  • concentration gradient wants to push ions out
  • inside of cell becomes less positive, outside becomes more positive –> creates electrical gradient
  • eventually will stop pushing them out and start pushing back in
  • when the 2 forces are equal = equilibrium potential
  • occurs at -75mv
25
Q

Nernst equation and Goldman equation

A

Nernst: (58/z)*log([Xo]/[Xi])

z = valence of ion

Goldman: not gonna type it out, but P = relative permeability at rest
Pk : PNa : PCl = 1.0 : 0.04 : 0.45

26
Q

resting membrane potential

  • 3 major ions
  • Na eq potential
  • permeability of Na and K
  • membrane potential result
  • chloride ions
A
  • 3 major ions: K+, Na+, Cl-
  • Na eq potential = +55mV
  • 2 forces on Na+ in –> conc and elec
  • membrane much less permeable to Na, but K+ has leak channels
  • Na has VERY FEW leak channels –> small conductance depolarizes cell a little –> results in Vm of -65mV instead of -75mV

chloride ions: much higher outside than inside

  • conc gradient drive Cl in, electro pushes out (since cell is slightly negative)
  • eq potential ~70mV
27
Q

sodium-potassium pump

A

3Na out, 2K in, against their electrochenical gradients

  • prevents mV from going to 0 (maintains gradients)
28
Q

3 stages of voltage-gated ion channels

A
  1. closed: least energy; during resting potential –> ball and chain inactivation gate not used
    - there is a positive chunk of protein which is attracted to the negative charge inside the cell –> keeps channel closed
    2: channel opens: inside becomes more positive, which causes change in conformation
    - when AP happens it gets repelled by the influx of positive charge and causes the gate to stay open
  2. ball and chain reacts to depol too, just slower –> closes gate and stops more Na ions from passing through
    - concentration of Na does not change much! but enough passes through to change the inside voltage of cell and create potential diff
    - will stay inactivated until cell is repolarized (refractory period)
29
Q

path of an action potential

A
  1. starts off at -65mV for RMP
  2. random EPSPs will occur, if one is big enough it will pass -55mV, which is the threshold value for starting an AP
  3. once passed threshold, Na voltage channels will open and influx of Na causes rapid depol –> Vm = ~40mV
  4. once at peak, Na channels will close, but K+ channels are open –> K+ ions flow out
  5. K+ channels close slowly –> causes hyperpolarization
  6. pump brings them back to RMP
30
Q

AP 1-way conduction

A
  • refractory period: even tho current flows passively in both directions, the AP can only travel in one direction since VG channels downstream are the only ones that can open due to the refractory period of the recently activated ones
31
Q

Hodgkin and Huxley model

- needs (3)

A

needs:
1. healthy cells
- in vitro –> culture/expression
- in vivo –> an acute slice or a live animal
2. microscope, electrodes, noise reduction (vibration dampening)
3. signal acquisition device –> amplifier, computer software

32
Q

Ohm’s Law

A

delta V = IR

V = voltage
I = current: flow of positive ions (Na+ rush in)
R = resistance: measured in Ohms --> anything that impedes flow of current

note: G = conductance (opposite of resistance) –> anything that allows/helps flow of current
G = 1/R

33
Q

electrophysiology experiment

  1. purpose of set up
  2. battery
  3. voltmeter
  4. controller
    - negative feedback loop
A
  1. Purpose of set up is to keep voltage constant while measuring current —> how current flows at diff potentials (voltage clamped at diff values)
    - Ohms law = voltage = current x resistance
    - If we can figure out two variables so we can figure out conductance (G = 1/R)
  2. We have battery/signal generator —> can set it to the voltage u want —> eg. -50mV
    - If u change membrane potential current also changes —> electrochemical forces/conductance
  3. Voltmeter tracks what is happening at the membrane
    - When you turn on system and change potential currents will change at membrane = changes membrane potential (diff concentration of ions) —> voltmeter measures change at membrane level
  4. Controller compares changes to what preset membrane potential was
    - If the potentials don’t match, controller will inject/remove current to bring back to OG —> counteracting the current to keep it at same voltage
    - negative feedback loop
    - We measure that injection/removal of current using internal current electrode (were not measuring the current itself, were measuring the amount of current required to keep the system at the same level –> compensatory current)
34
Q

current graphs

  • independent vs dependent
  • change is called?
  • Ic and Ll
  • small depol vs large depol
A

indep: voltage
dep: current
change = step
Ic = big jump at beginning (not important, always happens at beginning and end)
Ll = leaky current = steady outflow

small depol: inc voltage from -65 to -50mV –> causes a slight outward current –> the inside of cell becomes a bit more positive (due to EPSPs) which results in a slight outward current to bring back to RMP (above black dotted line = outward flow)

big depol: looks like an inverted AP –> directions are opposite of AP (when it goes down, it means the current is flowing into the cell, when it goes up, its flowing out of cell)

  • flow of current goes down and reaches “peak” –> inc in conductance (steep curve)
  • Na channels open (steep down); K channels open (counteract Na flow –> slow back up)
35
Q

voltage clamp recording

  • blocking ion channels –> TTX vs TEA
  • selectivity
  • K+ vs Na+ and current
A
  • can selectively block ion channels
  • tetrodotoxin (TTX)/Saxitoxin = blocks VG Na+ channels –> very toxic, cannot fire APs
  • tetraethylammonium (TEA) = blocks VG K+ channels –> less toxic, messes with some K+ channels
  • able to selectively look at one or the other channel by blocking the other
  • K lets current out of cell, Na lets current into cell
36
Q

Na+ and current

  • chemical driving force
  • electrical deriving force
  • net driving force
A
  • chemical driving force for current will always be strongly inward –> concentration of Na+ is so much greater outside the cell that the movement of ions will not be great enough to change that
  • electrical driving force for current is strongly outwards when the inside of the cell is positive, and strongly inwards when the inside of the cell is negative
  • therefore, net driving force is very strong inwards when inside of cell is negative, since both chemical and electrical are pushing inwards

*note: at 55mV the net driving force for Na+ is zero because that is its Eq voltage

37
Q

K+ and current

  • chemical driving force
  • electrical deriving force
  • net driving force
A
  • chemical driving force for current will always be strongly outward –> K+ is highly concentrated inside the cell –> global concentration will not change by the movement of ions
  • electrical driving force for current is outwards when the inside of the cell is positive, and inwards when the inside of the cell is negative
  • therefore, the net driving force is very strong outwards when inside of cell is positive, since both chemical and electrical are pushing outwards
38
Q

synaptic transmission

  • sherringham
  • renton
  • dale
  • loewi
  • eccles
A

Sherringham = synapse

Renton = (Adrenaline) —> EPI-like substance

  • Response of tissue mimics SNS activity –> chemical system for SNS?
  • Most ppl thought synapses communicated electrically (chemical too slow)

Dale = Ach —> mimics the activity of PNS —> something happening in the periphery

Loewi = frog heart expt

  • Live frog heart in bath, 2nd heart in same bath
  • Stimulates vagus nerve —> slows down heartbeat —> when he slowed one heart, second heart also slowed down

Eccles = activated inhibitory interneurons and measured activity in motor neurons
- they were hyperpolarizing motor neurons —> therefore it must be chemical

39
Q

synapse

  • chemical (+ advantages)
  • electrical (+ advantages)
A

chemical synapse: injected current causes an AP in the presyn cell –> release of NTs and change in V of the postsyn cell

  • more versatile, diff types of NTs w diff actions
  • can cover a large area, longer lasting effects
  • can manipulate –> inhibition patterns

electrical synapse: transmit signals btwn neurons –> current enters the postsyn cell through a gap junction channel

  • current can flow straight into next cell –> sharing cytoplasm
  • faster than chemical –> instantaneous
40
Q

visualizing vesicles during synaptic transmission

  • at rest
  • depol
  • exocytosis
  • endocytosis
A
  • at rest, vesicles are docked and ready to be released
  • stimulate a depol and flash freeze the tissue –> see what happens 5 ms after
  • can see fusion of vesicles with the presyn membrane (exocytosis)
  • 10 s later you can see endocytosis of vesicles –> vesicle recycles back –> filled with NTs or broken down in the soma
41
Q

excitatory neurotransmitters

  • NT
  • receptor
  • non-selective meaning
  • Na vs K during depol
  • reversal potential
  • postsynaptic result
A

glutamate: primary excitatory NT
AMPA receptors: glutamate ligand-gated ionotropic receptors
- non-selective –> both Na and K can pass through (cations)
- excitatory –> causes depol of the cell –> Na enters, K exits
- reversal potential: net current switches from in to out or vice versa
- excitatory NTs cause depol –> EPSP

42
Q

ionotropic inhibitory NT receptors

  • NT
  • receptor
  • causes what?
  • effect on RMP vs not RMP
  • clamping
  • firing rate
A

GABA: primary fast inhibitory NT

  • activated Cl- receptor channels (GABAa) (no net movement) but when EPSP happens at the same time as IPSP they cancel each other out
  • clamps membrane at -70mV –> responds to any depol and brings it back down –> prevents APs
  • alters firing rate of neurons by changing freq at which APs happen (changes electrical firing code)
43
Q

patch clamp recording

  • set up
  • goal
  • solution in micropipette
  • Ach
  • current
A

assess current in a very small region of membrane

  • form a seal with a glass micropipette by suctioning in (with electrode) and feed the membrane
  • goal is to have only 1 channel in the micropipette
  • solution mimics ECM –> can change concentration and see what happens
  • add Ach for muscle cells –> causes contraction –> channel opens for a bit –> able to get readings from electrode
44
Q

post-synaptic current recording

  • 2 things control EPSP
  • current
  • timing
A

2 things control EPSP

  1. length of time channels are open
  2. how many channels are bound
  • diff channels are activated by an Ach pulse –> each channel lets in a set amt of current (i, 2i, 3i, etc)
  • same current, but channels are open for diff amts of time –> can see a combination of all the currents on the electrode reading –> shows that the sum of the multiple currents is what you see in these experiments (same current speed, but diff amts of time)
45
Q

NT release is additive

A

weak stimulation to axons in a calcium-deficient env –> measure how big the depol is from a single vesicle at post syn

goal: make it so that 1 AP causes the release of 1 vesicle –> see the effect of 1 vesicle worth of NTs (try to get as close to the threshold as possible so that only 1 AP occurs)
- since its low Ca env, its less likely for vesicles to be released
- measure EPSP amplitude (mV) in post syn –> estimate how big of a depol from a single vesicle (becomes 1 unit of EPSP –> releases 1 vesicle)
- multimodal –> each mode is a multiple of 1 unit (eg. 0.4, 0.8, 0.12, etc)

question: how many vesicles are needed to fire an AP?
RMP = -65mV
threshold = -55 mV

-65 - (-55) = 10mV difference

10/u = x –> you need x amount of vesicles to fire an AP

u = unit value

46
Q

Brainbow

A

expression of multiple different coloured FPs
- randomly expressed FPs combine to create unique colours in different neurons

goal: track dendrites and axons of many or all neurons
- -> see how they connect and form circuits

47
Q

GRASP

A

GFP reconstitution across synaptic partners

  • fluorescent labeling of “synaptically connected” neurons
  • based on spatial proximity
  • promoters are key! –> label presynaptic machinery with complimenting “halves” of GFP

PSYC 370 (3 decks)

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